The present invention relates to methods for analysing one or more samples in an array of samples, preferably biomolecules, and an apparatus for carrying out these methods.
Improvements in laboratory techniques and practices have led to the discovery of an ever increasing number of new biomolecules. New protein purification and detection methods, for example, have allowed the detection of many possibly new proteins. Due to the large number of known biomolecules, it is now necessary to carry out molecular comparisons of newly discovered molecules to determine to what extent they are similar to or different from known molecules. To carry out definitive analyses for proteins for example it is necessary to obtain amino acid sequence information. Unfortunately, current methods and apparatus for such analyses are slow and are only able to analyse one or a few samples at one time. In order to carry out analysis of a given protein at present it is necessary to obtain the protein in substantially pure and isolated form. There is a need for methods and apparatus that can analyse one desired sample from an array of samples or be able to analyse multiple samples.
The present inventors have now realised that it is possible to develop improved methods and apparatus suitable to carry out these types of analyses.
The present invention relates generally to methods for analysing at least one sample in an array of samples by recording an image of the position of at least one sample relative to the other samples in the array and utilizing the recorded image so as to allow the analysis of the at least one sample in situ.
In a first aspect, the present invention consists of a method for analysing at least one sample in an array of samples, the method including the steps:
(a) recording an image of the position of at least one sample relative to the other samples in the array;
(b) utilizing the recorded image so as to allow the application of a reagent or a succession of reagents to the at least one sample in situ; and
(c) analysing the at least one sample for a reaction to or with the reagent(s).
In a preferred form, the samples are biomolecules selected from the group consisting of proteins, peptides, saccharides, lipids, nucleic acid molecules, complex biomolecules including glycoproteins, and mixtures thereof. The biomolecules are preferably separated by chromatography to form an array of samples. The chromatography is preferably electrophoresis, and more preferably electrophoresis is carried out in a polyacrylamide gel.
The polyacrylamide gel electrophoresis can be carried out in one dimension including isoelectric focusing, native polyacrylamide gel electrophoresis, and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Alternatively, the polyacrylamide gel electrophoresis is carried out in two dimensions with the first dimension by isoelectric focusing and the second dimension is by SDS polyacrylamide gel electrophoresis.
Preferably, the biomolecules separated by electrophoresis are transferred to a semi-solid or solid support. The solid support can be a membrane made of polyvinylidene difluoride, nitrocellulose, nylon, Teflon(trademark), Zitex(trademark), polypropylene, polytetrafluoroethylene, and derivatised forms thereof having one or more functional groups.
Preferably, the biomolecules transferred to semi-solid or solid support are visualised by association with a dye, fluorescent group or metal, or by association with a second biomolecule which is coupled with a third biomolecule, dye, fluorescent group or metal. The array of samples is preferably in a plane in order to assist in the recording of the image.
In a preferred embodiment of the present invention, the image is generated from a scan of the samples stained or illuminated to allow them to be visualised and the application of the reagent or reagents is carried out using a chemical printer based on an xe2x80x9cink jetxe2x80x9d or similar application system where the reagent or reagents are discharged to the desired sample by the chemical printer.
It will be appreciated that the array to be manipulated may not necessarily be the array from which the image was obtained. For example, it would be possible to make multiple identical arrays of samples and use one array to obtain the image but use one or more of the multiple identical arrays to carry out the manipulations. For example in protein separation by 2 dimension polyacrylamide gel electrophoresis (2D PAGE) one separation gel may be blotted to more than one support or multiple identical separations carried out and each transferred to a support to form the identical arrays.
The analysing may be by any means known to the art. Suitable examples include use of liquid chromatograph, photoelectrical, photochemical, laser, radiochemical, and mass spectral analyses. The sample may be analysed directly for a given reaction product. Alternatively, where reagent has been applied to one sample in the array and has reacted with the one of the sample treated, it would be possible to analyse the array of samples and the detection of a reaction product would be assigned as being derived from the one sample treated.
The image can be recorded by any suitable means including recorded as an electronic or digital image. In one preferred form, the image is generated from a scan of the samples stained or illuminated to allow the samples to be visualised and the application of the reagent or reagents is carried out using a chemical printer application system where the reagent or reagents are discharged to the desired sample by the chemical printer.
It will be appreciated that steps (a) and (b) can be repeated or cycled so as to carry out a series of manipulations of the same sample or a number of different samples in the array. The multiple manipulations can be with the same reagent, the same set of reagents, or a number of different reagents.
The analysing is preferably by liquid chromatography, photoelectrical, photochemical, laser, radiochemical, or mass spectral analyses. It will be appreciated that the sample can be analysed directly for a given reaction product.
In order to generate an image of the samples in the array, it is usually necessary to make them identifiable in some manner. Labelling the samples with a visible marker is one example that would allow the visualisation of the position of the samples with a charged coupled device (CCD). A scan of the labelled samples would then be recorded digitally and stored in a computer for example. Once the image has been recorded in digital form for example, there would be no need to maintain the visualisation of the samples on the array as the image is maintained electronically. If the locations of the samples are recorded on an X/Y grid, this would be one way of accessing the positions of the samples electronically or digitally. The computer would also control the application of the reagent in step (b) to the position of the sample to be manipulated. The amount of chemical delivered to a sample would be regulated in the same manner as grey-scale for black and white printing. The position of all the samples would be known from their co-ordinates on the grid, for example, and so further manipulation is possible regardless of whether or not the samples are still visible.
The present invention is particularly suitable for the multiple analyses of one or more samples, particularly on an array like a protein blot. The method is applicable for N- and C-terminal determination of proteins and peptides derived from proteins separated by, for example, two dimensional gel electrophoresis. It will be appreciated that the method can be used for antibody or antigen assays of multiple samples and the like and the present method makes possible the different subsequent steps based on the outcome of an earlier reaction.
In one preferred form, the invention concerns the development of a chemical printer which sprays a chemical reagent to a sample which has been absorbed onto a solid support so as to cause a detectable reaction with the molecules in the sample. The analysis of a chemical derivative of the molecules of the sample can be by laser desorption ionisation while it is still absorbed on the support. Furthermore, it is possible to analyse a different part of the same sample following each cycle of chemistry since the laser desorption technology can be aimed accurately at different regions of the sample.
A suitable chemical printing system for use in the present invention involves the use of piezoelectric drop-on-demand ink-jet printing technology for microdispensing fluids in DNA diagnostics or the Combion, Inc. synthesis process called xe2x80x9cChem-jetxe2x80x9d. To explore drop-on-demand fluid dispensing for DNA diagnostics, an eight fluid printer has been developed as part of the Genosensor Technology Development (GTD) project funded by the National Institute of Standards and Technology (USA). Research to date has focused on xe2x80x9cprintingxe2x80x9d oligonucleotide microspots onto solid supports. In the xe2x80x9cChem-jetxe2x80x9d technique, which was developed at the California Institute of Technology, tiny volumes of reagent-bearing liquid are squirted onto specific spots, or addresses, of a solid substrate much as an ink-jet printer squirts tiny dots onto a page. By repeatedly returning to each address with one or another of a small set of building blocks, in this case nucleotides modified for the process, huge two-dimensional libraries of short DNA chains (oligonucleotides) can be assembled.
The GTD and xe2x80x9cChem-jetxe2x80x9d are sensor/synthesis instruments rather than analysers. Hence, the present invention embraces a new application for the ink jet assembly technology.
It will be appreciated that many different chemistries can be performed in situ on samples on supports, particularly for proteins, in light of the recent developments of micropreparative immobilised pH gradient isoelectric focusing. Laboratory examples include the enzymic digests of visualised protein spots, the enzymic and chemical release of oligosaccharides attached to stained protein spots, and antibody/antigen reactions. It will be appreciated that such analyses can be conducted sequentially by the chemical printer. A further application of the chemical printer is large scale Edman chemistry and C-terminal chemistry on all proteins separated by two-dimensional electrophoresis.
Many additional permutations of the printer are possible:
i) The analysis of peptides after endoproteinase digests of a single (or small number) of proteins where, following a cycle of Edman chemistry, the released amino acids from each cycle of chemistry are analysed using laser desorption TOF-MS in combination with bioinformatics approach to identify the protein.
ii) Other macromolecules, for example complex carbohydrates and lipids which have been separated on thin layer chromatography or other supports.
iii) For blocked proteins, detected by absence of an amino acid after printing the first cycle of Edman chemistry, the support is then printed with an endoproteinase. Following the digest, the support is then subjected to a xe2x80x98printingxe2x80x99 of an end group blocking reagent which is specific for a particular sequence motif. For example, following an endoproteinase Asp-N digestion and one cycle (or xe2x80x98printingxe2x80x99) of Edman chemistry, the support is printed with o-phthalaldehyde (OPA), which blocks all xcex1-amino groups leaving only peptides with an N-terminal Pro. Hence, the process is specific for the motif Asp-Pro. Alternatively, following the trypsin digest, a cycle of Edman chemistry and a printing of OPA, the process is specific for the motif Lys/Arg-Xaa-Pro. These unblocked peptides can be analysed according to (i) above.
In a second aspect, the present invention consists in an apparatus for carrying out the first aspect of the present invention.
An apparatus for analysing at least one sample in an array of samples, the apparatus including:
(a) means for recording an image of the position of the at least one sample relative to the other samples in the array;
(b) means for applying a reagent or sequence of reagents to the at least one sample in situ;
(c) means for analysing the at least one sample for a reaction to or with the reagent(s); and
(d) control means for means (b), wherein means (b) applies the reagent to the at least one sample according to the position of the sample relative to the other samples in the array determined by means (a).
The apparatus may further include means for recording the analysis results obtained by means (c).
Preferably, the means (a) is selected from the group consisting of scanner, photodetector, and charged coupled device.
Means (b) may be a chemical printer adapted to apply one or more reagents to the sample. Alternatively, means (b) is a reagent delivery and extraction device including a fluid source, a fluid control means, a fluid delivery and sampling device for delivering fluid to a sample in the array and for sampling fluid applied to the sample on the array, and an extraction device capable of retaining a reaction product from a sample on the array; a control device for controlling fluid movement from the fluid source to the fluid delivery and sampling device and for controlling fluid movement from the sample to the extraction device via the fluid delivery and sampling device; wherein, in use, fluid capable of producing a reaction product from a sample is applied to a sample on the array from the fluid source via the fluid control means to the fluid delivery and sampling device by the action of the control device, and wherein a portion of the fluid applied to the sample is drawn through the fluid delivery and sampling device to the extraction device by the action of the control device.
The extraction device is not necessary for the present invention but may be useful when certain analyses of treated samples are required. For example, the extraction device is particularly suitable for introducing a sample into an electrospray mass spectrometer. The extraction device, however, may not be necessary for introducing the sample into the MALDI-TOF as this instrument is much more tolerant of salts.
Preferably the fluid control means is a 3-way valve.
Preferably, the control device comprises a first control device for the fluid delivery and sampling device and a second control device for the extraction device. More preferably, the first and second control devices are piezoelectric control devices.
The fluid is preferably a reagent capable of reacting with a sample to produce a detectable product capable of being retained by the extraction device. The extraction device is preferably a cartridge of chromatography packing.
In a preferred form means (c) is selected from the group consisting of liquid chromatograph, photoelectrical detector, photochemical detector, laser detector, radiochemical detector, and mass spectrometer. Means (d) can be a computer programmed to control means (b).
The array of samples is preferably on a semi-solid or substantially solid support. One advantage of the present invention is that very small samples may be treated and analysed. The apparatus according to the present invention is particularly suitable for analysing samples having an area less than about 100 mM2, preferably less than about 50 mm2, more preferably about 1 to 10 mm2. It will be appreciated, however, that even smaller samples may be analysed by the present invention.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and accompanying drawings.